Type of Thesis
X-Chromosome silencing is a highly conserved process that is essential for proper gene dosage in female mammalian cells. In humans, the process is initiated early in embryonic development and will persist throughout the organism’s life. Improper gene dosage is manifest in disorders such as Down Syndrome, but in the context of X chromosome inactivation, it is fatal. The process occurs within the nucleus of cells, often on the nuclear lamina, and is facilitated by a long non-coding RNA called Xist. Xist, which stands for X-inactivation specific transcript, works together with a suite of proteins that are involved with and implicated in the entire chromosomal silencing process. Xist coats the future silenced chromosome in cis and along with its various protein partners, it is responsible for condensing the X chromosome into what is colloquially called a Barr body on the nuclear edge.
Because the presence of Xist RNA is essential for X-chromosome inactivation and Xist, in turn, depends on various protein partners for proper function, identifying proteins that directly interact with Xist will provide insights into mechanisms that dictate this process. A suite of in vivo studies has identified proteins that directly interact with Xist through cross-linking pull-down methods. One of the proteins required for the silencing of the X-chromosome is SHARP (SMRT/NCoR HDAC Associated Repressor Protein), a 407 kDa nuclear co-repressor protein that consists of 7 domains including several RRMs that are involved in targeting SHARP to Xist. Transcriptional silencing is proposed/believed to occur through the recruitment of SHARP to Xist, followed by recruitment of HDAC3, which excludes RNA polymerase II, and the subsequent recruitment of PRC2 and its follow-on repressive mechanism of methyltransferase. More significantly, studies have shown that knocking out SHARP abrogates Xist’s ability to silence the X-chromosome.
The goal of my thesis project has been to better elucidate this essential interaction between Xist RNA and SHARP. Our results suggest that this vital biological function relies upon specific structural motifs comprised of key nucleotide sequences that serve as the platform for SHARP recruitment by the A repeat region of Xist.
Averick, Torey, "Characterizing the interaction between the A-repeat region of Xist and SHARP" (2018). Undergraduate Honors Theses. 1662.